In animals, proteinases are important in wound healing, extracellular matrix destruction, tissue reorganization, and in cascades leading to blood coagulation, fibrinolysis, and complement activation. Proteinases are released by inflammatory cells for destruction of pathogens or foreign materials, and by normal and cancerous cells as they move through their surroundings. Overproduction or lack of regulation of proteinases can also have pathological consequences. Elastase, released within the lung in response to the presence of foreign particles, can damage lung tissue if its activity is not tightly regulated. Emphysema in smokers is believed to arise from an imbalance between elastase and its inhibitor, alpha-1-antitrypsin. This balance may be restored by administration of exogenous alpha-1-antitrypsin.
Proteinases are also used within a wide range of applications in industry and research. Through the use of proteinases and other enzymes, industrial processes can be carried out at reduced temperatures and pressures and with less dependence on the use of corrosive or toxic substances. The use of enzymes can thus reduce production costs, energy consumption, and pollution as compared to non-enzymatic products and processes. Industrial applications of proteinases include food processing, brewing, and alcohol production. Proteinases are important components of laundry detergents and other products. Within biological research, proteinases are used in purification processes to degrade unwanted proteins.
The activity of proteinases is regulated by inhibitors; 10% of the proteins in blood serum are proteinase inhibitors (Roberts et al., Critical Reviews in Eukaryotic Gene Expression 5:385-436, 1995). One family of proteinase inhibitors, the serpins (serine proteinase inhibitors), includes inhibitors of elastase, trypsin, chymotrypsin, thrombin, plasmin, and other proteinases. These inhibitors thus regulate a variety of physiological processes, including blood coagulation, fibrinolysis, complement activation, inflammation, and tumor development.
Proteinase inhibitors regulate the proteolytic activity of target proteinases by occupying the active site and thereby preventing occupation by normal substrates. The serpins possess an exposed loop (variously termed an “inhibitor loop”, a “reactive core”, a “reactive site”, or a “binding loop”) that is accessible to the target proteinase. Interaction between inhibitor and enzyme produces a stable complex which disassociates very slowly, releasing a modified inhibitor that is cleaved at the scissile bond of the binding loop (the P1-P1′ bond). See, Carrell et al., Cold Spring Harbor Symp. Quant. Biol. LII:527-535, 1987.
Serpins are characterized by a highly ordered, globular structure composed of pleated sheets and α helices. Although serpins share the same overall tertiary structure, they are quite diverse in sequence. Even closely related members of this family may be only about 30% identical in amino acid sequence (Carrell et al., ibid.). Inhibitory specificity is determined primarily by the identities of the amino acid residues in the P1-P2 positions.
One subfamily of serpins are structurally related to ovalbumin. These “ov-serpins” lack cleavable secretory peptides, and many function intracellularly. Some members of this subfamily are involved in the regulation of inflammation and apoptosis. This subfamily includes the interleukin-1β converting enzyme (ICE) inhibitor crmA from cowpox virus (Ray et al., Cell 69:597-604, 1992) and CAP-3 (Sprecher, U.S. Pat. No. 5,747,645). Some members of this subfamily, including ovalbumin, plasminogen activator inhibitor-2 (PAI-2), squamous cell carcinoma antigen (SCCA), and maspin, are efficiently secreted, apparently due to an internal secretion signal. PAI-2 may also function intracellularly. The intracellular Ov-serpins have been reviewed by Korpula-Mastalerz and Dubin (Acta Biochim. Polonica 43:419-430, 1996).
In view of the specificity of proteolytic enzymes and the growing use of proteinases in industry, research, and medicine, there is an ongoing need in the art for new enzymes and new enzyme inhibitors. The present invention addresses these needs and provides other, related advantages.